Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive damage to the joints. Inflammation of peripheral joints occurs (e.g. in wrists and metacarpophalangeal joints) and often results in progressive destruction of articular structures, which is usually accompanied by systemic symptoms. RA affects about 1% of the population, with women affected 2 to 3 times more often than men. Onset may be at any age, most often between 35 and 50 years, although children or the elderly can be affected as well.
The disease is characterized by an abnormal growth of connective tissue in the joints, including synovial tissue. Damage has been shown to be mediated by cytokines, chemokines, and metalloproteases. The joints of chronic rheumatoid arthritis patients have marked growth of synovial cells, formation of a multilayer structure due to abnormal growth of the synovial cells (pannus formation), invasion of the synovial cells into cartilage tissue and bone tissue, vascularization toward the synovial tissue, and infiltration of inflammatory cells such as lymphocytes and macrophages. Mechanisms of onset of chronic rheumatoid arthritis have been reported to be based on such factors as heredity, bacterial infection and the contribution of various cytokines and growth factors, but the overall mechanism of onset has remained unclear.
Although attempts have been made to develop therapeutic agents for treating RA, there is at present no satisfactory cure for the disease. In addition, many of the therapeutic agents administered to alleviate pain and inflammation associated with the disease, such as disease-modifying anti-rheumatic drugs (DMARDs) and non-steroidal anti-inflammatory agents (NSAIDs), produce intolerable side effects.
NSAIDs are of some help for alleviating the pain of RA but do not prevent erosions or disease progression. NSAIDs can cause peptic ulcer disease and gastrointestinal bleeding. Other possible adverse effects include headache, confusion and other neurological symptoms, worsening of hypertension, edema, and decreased platelet adhesiveness. Cyclooxygenase-2 (COX-2) inhibitors, or coxibs (e.g., celecoxib), appear to have efficacy comparable to nonselective NSAIDs and are less likely to cause gastrointestinal toxicity. However, recent evidence indicates that some, if not all, coxibs increase the risk of cardiovascular events (e.g. stroke) with long-term use.
Disease-modifying anti-rheumatic drugs (DMARDs) appear to slow the progression of RA and are indicated in nearly all patients with RA. About ⅔ of patients improve overall, but complete remissions are uncommon. They have minimal immediate analgesic effects, so NSAIDs must often be continued. Examples of DMARDs include methotrexate, hydroxychloroquine, sulfasalazine, parenteral gold compounds, and oral penicillamine.
Systemic corticosteroids decrease symptoms more rapidly and to a greater degree than other drugs. However, they do not prevent joint destruction, and their clinical benefit often diminishes with time. Furthermore, severe rebound follows the withdrawal of corticosteroids in active disease. Because of their long-term adverse effects, many doctors recommend that corticosteroids be given to maintain function only until another DMARD has taken effect.
Cytotoxic or immunosuppressive drugs (e.g. azathioprine, cyclosporine and cyclophosphamide) provides efficacy similar to DMARDs. However, immunosuppressants are more toxic, particularly cyclophosphamide, which can cause bone marrow suppression and increase risk of cancer. Thus, these drugs are used only for patients in whom treatment with DMARDs has failed or to decrease the need for corticosteroids.
Other agents, such as interleukin-1 (IL-1) receptor antagonists and TNF-α antagonists, are also used for the treatment of RA in adults, often in combination with other treatments. Common side effects include cytopenia and infection.
However, the need remains for effective treatments useful for treating RA. Recently, attempts to develop specific nucleic acid agents (such as antisense and RNA interfering molecules) that may potentially be used in the therapy of RA have been reported. For example, PCT Pub. Nos. WO 2005/112971, WO 2005/079862, and WO 03/070897 disclose downregulating nucleic acid agents directed to proprotein convertase, resistin and TNF alpha, respectively. However, currently no nucleic acid based agent is in use for treating rheumatoid arthritis in humans.
Silencing Oligonucleic Acids
The silencing or down regulation of specific gene expression in a cell can be effected by oligonucleic acids using techniques known as antisense therapy, RNA interference (RNAi), and enzymatic nucleic acid molecules.
Antisense therapy refers to the process of inactivating target DNA or mRNA sequences through the use of complementary DNA or RNA oligonucleic acids, thereby inhibiting gene transcription or translation. An antisense molecule can be single stranded, double stranded or triple helix.
Other agents capable of inhibiting expression are for example enzymatic nucleic acid molecules such as DNAzymes and ribozymes, capable of specifically cleaving an mRNA transcript of interest. DNAzymes are single-stranded deoxyribonucleotides that are capable of cleaving both single- and double-stranded target sequences. Ribozymes are catalytic ribonucleic acid molecules that are increasingly being used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest.
RNA interference (hereinafter “RNAi”) is a method of post-transcriptional inhibition of gene expression that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide) double stranded RNA (“dsRNA”) molecules, which are present in the cell. These short dsRNA molecules, called “short interfering RNA” or “siRNA”, cause the destruction of messenger RNAs (“mRNAs”) that share sequence homology with the siRNA to within one nucleotide resolution. It is believed that the siRNA and the targeted mRNA bind to an “RNA-induced silencing complex” or “RISC”, which cleaves the targeted mRNA. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
U.S. Pat. No. 6,506,559 to Fire et al. teaches genetic inhibition by double-stranded RNA, particularly a process for inhibition of gene expression of a target gene in a cell using RNA having a region with double-stranded structure, wherein the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical.
PCT Pub. No. WO 01/75164 to Tuschl et al. discloses that synthetic siRNA of 21 and 22 nucleotides in length, and which have short 3′ overhangs, are able to induce RNAi of target mRNA in a Drosophila cell lysate. Cultured mammalian cells also exhibit RNAi degradation with synthetic siRNA. PCT Pub. No. WO 02/44321 relates to sequence and structural features of double-stranded (ds) RNA molecules required to mediate target-specific nucleic acid modifications such as RNA-interference and/or DNA methylation.
PCT Pub. No. WO 2006/060454 teaches methods of designing small interfering RNAs, antisense polynucleotides, and other hybridizing nucleotides. US Patent Application Publication No. 2006/0217331 discloses chemically modified double stranded nucleic acid molecules for RNA interference.
H19 and Use Thereof in Cancer Diagnosis and Therapy
H19 was the first human imprinted non protein-coding gene to be identified showing expression of only the maternal allele. It is also imprinted in mice. H19 was mapped on the short arm of the human chromosome 11, band 15.5, homologous to a region of murine chromosome 7. It belongs to a group of genes that very likely does not code for a protein product. H19 gene is abundantly expressed in embryogenesis but is shut off in most tissues after birth. However, studies of various tumors have demonstrated a re-expression or an over-expression of the H19 gene when compared to healthy tissues. Moreover in cancers of different etiologies and lineages, aberrant expression in allelic pattern was observed in some cases. While H19 shows mono-allelic expression in most tissues throughout development, with the exception of germ cells at certain stages of maturation, and in extra-villous trophoblasts, bi-allelic expression of this gene, referred as “relaxation of imprinting” or “loss of imprinting”, have been found in an increasing number of cancers, for example, hepatocellular carcinoma, liver neoplasms, lung adenocarcinoma, esophageal, ovarian, rhabdomyosarcoma, cervical, bladder, head and neck squamous cell carcinoma, colorectal, uterus and in testicular germ cell tumors. Today nearly 30 types of cancers show dysregulated expression of H19 gene as compared to healthy tissues, with or without loss of imprinting.
Gene expression analyses using cancer cell lines have identified a plethora of downstream effectors of H19 RNA. Among these are group of genes that were previously reported to play crucial roles in some aspects of the tumorigenic process (Ayesh et al., 2002; Matouk et al., 2007; Lottin et al., 2002). H19 RNA presence may enhance the invasive, migratory and angiogenic capacity of the cell by up regulating genes that function in those pathways, and thus could contribute at least to the initial steps of the metastatic cascade. Additional studies highlight the potential role of H19 in promoting cancer progression and tumor metastasis by being a gene responsive to HGF/SF.
The specific expression of H19 gene in cancer cells has prompted its use in clinical applications for diagnosing cancer. For example, U.S. Pat. No. 5,955,273 to some of the inventors of the present invention teaches the use of H19 gene as a tumor specific marker. PCT Pub. No. WO 2004/024957 to some of the inventors of the present invention discloses the use of H19 for the detection, in a patient suspected of having cancer, of the presence of residual cancer cells or micro-metastases originating from solid tumors.
PCT Pub. No. WO 99/18195 teaches the specific expression of heterologous sequences, particularly genes encoding cytotoxic products (e.g. Diphtheria toxin), in tumor cells under the control of cancer specific promoters (e.g., H19 promoter).
A publication by Stuhlmúller et al. (2003) discloses that H19 RNA is expressed in RA synovial tissue. The Stuhlmúller et al. publication demonstrates an increased expression of H19 in synovial fibroblasts grown in vitro under serum starvation conditions, and consequently postulates that H19 might have a pathogenic role in RA. According to Stuhlmúller et al., the pathophysiological role of H19 RNA remains elusive, and its particular role in RA awaits elucidation by functional studies and mutation analysis. Stuhlmúller et al. do not teach or suggest nucleic acid agents useful for treating RA.
PCT Pub. No. WO 04/031359 teaches a method for regulating the expression of angiogenesis-controlling genes in cells that are involved in neo-vascularization, comprising administering to the cells an effective amount of an H19 modulator. WO 04/031359 provides a list of angiogenesis-associated conditions, which purportedly may potentially be treated by either increasing or decreasing H19 expression, including, inter alia, RA. While a number of angiogenesis-associated genes were reported to be up-regulated in a carcinoma cell line transfected with an H19-expressing construct, down-regulation of H19 was not demonstrated. Specific and efficacious siRNA agents, capable of down-regulating H19, were neither taught nor suggested.
A publication by Berteaux et al. (2005) discloses two specific siRNA molecules targeted to H19, which arrest in vitro growth of breast cancer cells.
Additional species of siRNA intended for silencing H19 are now also available from commercial sources, including Invitrogen, Dharmacon and Qiagen. The efficacy of such commercially available H19 siRNA sequences is putative and their utility remains to be established. Certain commercially available molecules correspond to SEQ ID NOs: 14-25 of the present application.
WO 2007/034487 discloses a nucleic acid construct comprising: (i) a first nucleic acid sequence encoding TNF alpha; (ii) a second nucleic acid sequence encoding a Diphtheria toxin; and (iii) at least one additional nucleic acid sequence comprising a cancer specific promoter (e.g. an H19 promoter); the TNF alpha and Diphtheria toxin encoding sequences being under an expression control of the cancer specific promoter. Also provided are construct systems and methods and uses of same.
WO 2007/007317, published after the priority date of the present invention, discloses isolated oligonucleotides capable of down-regulating a level of H19 mRNA in cancer cells, corresponding to SEQ ID NOS: 5-8 of the present invention. Also disclosed are articles of manufacture comprising agents capable of downregulating H19 mRNA in combination with an additional anti-cancer treatment as well as methods of treating cancer by administering same.
None of the prior art discloses or suggests that nucleic acid agents that inhibit H19 expression may be applied effectively in rheumatoid arthritis therapy. There remains an unmet medical need for therapeutic modalities useful for treating rheumatoid arthritis and inhibiting symptoms associated therewith.